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International audience; An inorganic sol−gel polymerization process was used as a cross-linking reaction during three-dimensional (3D) bioprinting of cell-containing hydrogel scaffolds. Hybrid hydroxypropyl methyl cellulose (HPMC), with a controlled ratio of silylation, was prepared and isolated as a 3D-network precursor. When dissolved in a biological buffer containing human mesenchymal stem cells, it yields a bioink that can be printed during polymerization by extrusion. It is worth noting that the sol−gel process proceeded at pH 7.4 using biocompatible mode of catalysis (NaF and glycine). The printing window was determined by rheology and viscosity measurements. The physicochemical properties of hydrogels were studied. Covalent functionalization of the network can be easily performed by adding a triethoxysilyl-containing molecule; a fluorescent hybrid molecule was used as a proof of concept.

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International audience; Background: Systemic sclerosis (SSc) is a severe autoimmune disease for which mesenchymal stromal cells (MSCs)-based therapy was reported to reduce SSc-related symptoms in pre-clinical studies. Recently, extracellular vesicles released by MSCs (MSC-EVs) were shown to mediate most of their therapeutic effect. Here, we aimed at improving their efficacy by increasing the MSC-EV dose or by IFNγ-priming of MSCs.Methods: small size (ssEVs) and large size EVs (lsEVs) were recovered from murine MSCs that were pre-activated using 1 or 20 ng/mL of IFNγ. In the HOCl-induced model of SSc, mice were treated with EVs at day 21 and sacrificed at day 42. Lung and skin samples were collected for histological and molecular analyses.Results: increasing the dose of MSC-EVs did not add benefit to the dose previously reported to be efficient in SSc. By contrast, IFNγ pre-activation improved MSC-EVs-based treatment, essentially in the lungs. Low doses of IFNγ decreased the expression of fibrotic markers, while high doses improved remodeling and anti-inflammatory markers. IFNγ pre-activation upregulated iNos, IL1ra and Il6 in MSCs and ssEVs and the PGE2 protein in lsEVs.Conclusion: IFNγ-pre-activation improved the therapeutic effect of MSC-EVs preferentially in the lungs of SSc mice by modulating anti-inflammatory and anti-fibrotic markers.

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National audience; Background. Articular cartilage is a tissue with poor self-repair capacity, which is prone to progressive destruction after injuries. In cartilage, the chondrocytes surrounded by their extracellular matrix are organized in a complex structure that plays an essential weight-bearing role in the joint and can both sense and respond to various mechanical stimuli. Cartilage micropellet is a relevant and widely used in vitro model to study cartilage growth but poorly investigated in terms of mechanical characterization because of its small size and imperfect round shape. The objective of the study was to develop an original custom-made device allowing both the mechanical stimulation and characterization of mesenchymal stromal cells (MSCs)-derived cartilage micropellets. Methods. Alginate beads were made with a solution of 3 % sodium alginate extruded into 0,1 M CaCl2. Collagen microspheres were prepared with type I collagen in hydrofluoroether. Human bone marrow-derived MSCs were differentiated into chondrocytes by culture in micropellets with 10 ng/mL TGFβ3- containing inductive medium for 21 days. The fluidic-based device was designed for the concomitant culture of six micropellets placed into the conical wells of a chamber where they were stimulated by a positive pressure (sinusoidal, square or constant). The sinking of each micropellet into the cone and its deformation were recorded by a camera. Expression of the chondrocyte markers SOX9, AGG and COL2B were quantified 24 hours after stimulation by RT-qPCR. The Young’s modulus was determined using a finite element model employing a neo-Hookean hyperelastic law. Results. Alginate- and collagen-based microspheres were first used to validate the reliability of the device. Repeatability and reproducibility of pressure signals used for mechanical stimulation were demonstrated. The mechanical properties of the microspheres were equivalent to those determined by a conventional compression test and shown to be reproducible. MSC-derived cartilage micropellets were stimulated with sinusoidal, square or constant pressure. Different parameters (amplitude, frequency, duration) of the square pressure signal were tested on the expression of chondrocyte markers. A stimulation of 1 Hz at 7 kPa for 30 min induced a significant increase of chondrocyte markers. The mechanical properties of the micropellets were measured and a Young’s modulus of 47.6 ± 26.2 kPa was determined. Conclusion. The interest of this new device lies in the reliability to mechanically stimulate and characterize microspheres with radius in range of 600 to 1300 μm. Of importance in case of MSC-based cartilage micropellets, mechanical stimulation can be performed in parallel on six microspheres allowing the molecular and mechanical characterization on the same group of samples. In the future, the device will be useful to evaluate the growth of cartilage micropellets under mechanical stimuli in a longitudinal study.

International audience; Extracellular vesicles (EVs), including exosomes and microvesicles, derived from mesenchymal stem/stromal cells (MSCs) exert similar effects as their parental cells, and are of interest for various therapeutic applications. EVs can act through uptake by the target cells followed by release of their cargo inside the cytoplasm, or through interaction of membrane-bound ligands with receptors expressed on target cells to stimulate downstream intracellular pathways. EV-based therapeutics may be directly used as substitutes of intact cells or after modification for targeted drug delivery. However, for the development of EV-based therapeutics, several production, isolation, and characterization requirements have to be met and the quality of the final product has to be tested before its clinical implementation. In this review, we discuss the challenges associated with the development of EV-based therapeutics and the regulatory specifications for their successful clinical translation.

International audience; Background Articular cartilage is a tissue with poor self-repair capacity, which is prone to progressive destruction after injuries. In cartilage, the chondrocytes surrounded by their extracellular matrix are organized in a complex structure that plays an essential weight-bearing role in the joint and can both sense and respond to various mechanical stimuli. Cartilage micropellet is a relevant and widely used in vitro model to study cartilage growth but poorly investigated in terms of mechanical characterization because of its small size and imperfect round shape. The objective of the study was to develop an original custom-made device allowing both the mechanical stimulation and characterization of mesenchymal stromal cells (MSCs)-derived cartilage micropellets. Methods Human bone marrow-derived MSCs were differentiated into chondrocytes by culture in micropellets with 10 ng/mL TGFβ3-containing inductive medium for 21 days. The fluidic-based device was designed for the concomitant culture of six micropellets placed into the conical wells of a chamber where they were stimulated by a positive pressure (sinusoidal, square or constant). The sinking of each micropellet into the cone and its deformation were recorded by a camera. Expression of the chondrocyte markers SOX9, AGG and COL2B were quantified 24 hours after stimulation by RT-qPCR. The Young modulus were determined using a finite element model employing a neo-Hookean hyperelastic law. Results Alginate- and collagen-based microspheres were first used to validate the reliability of the device. Repeatability and reproducibility of pressure signals used for mechanical stimulation were demonstrated. The mechanical properties of the microspheres were equivalent to those determined by a conventional compression test and shown to be reproducible. MSC-derived cartilage micropellets were stimulated with sinusoidal, square or constant pressure. Different parameters (amplitude, frequency, duration) of the square pressure signal were tested on the expression of chondrocyte genes. A stimulation of 1 Hz at 0.07 bar during 30 min induced a significant increase of chondrocyte markers. The mechanical properties of the micropellets were measured and a Young’s modulus of 77.8 ± 47.8 kPa was determined. Conclusion The interest of this new device lies in the reliability to mechanically stimulate and characterize microspheres with radius in range of 600 to 1300 μm. Of importance in case of MSC-based cartilage micropellets, mechanical stimulation can be performed in parallel on six microspheres allowing the molecular and mechanical characterization on the same group of samples. In the future, the device will be useful to evaluate the growth of cartilage micropellets under mechanical stimuli in a longitudinal study.

International audience; Aging is associated with high prevalence of chronic degenerative diseases that take a large part of the increasing burden of morbidities in a growing demographic of elderly people. Aging is a complex process that involves cell autonomous and cell non-autonomous mechanisms where senescence plays an important role. Senescence is characterized by the loss of proliferative potential, resistance to cell death by apoptosis and expression of a senescence-associated secretory phenotype (SASP). SASP includes pro-inflammatory cytokines and chemokines, tissue-damaging proteases, growth factors; all contributing to tissue microenvironment alteration and loss of tissue homeostasis. Emerging evidence suggests that the changes in the number and composition of extracellular vesicles (EVs) released by senescent cells contribute to the adverse effects of senescence in aging. In addition, age-related alterations in mesenchymal stem/stromal cells (MSCs) have been associated to dysregulated functions. The loss of functional stem cells necessary to maintain tissue homeostasis likely directly contributes to aging. In this review, we will focus on the characteristics and role of EVs isolated from senescent MSCs, the potential effect of MSC-derived EVs in aging and discuss their therapeutic potential to improve age-related diseases.

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International audience; Tissue accumulation of p16INK4a-positive senescent cells is associated with age-related disorders, such as osteoarthritis (OA). These cell-cycle arrested cells affect tissue function through a specific secretory phenotype. The links between OA onset and senescence remain poorly described. Using experimental OA protocol and transgenic Cdkn2a+/luc and Cdkn2aluc/luc mice, we found that the senescence-driving p16INK4a is a marker of the disease, expressed by the synovial tissue, but is also an actor: its somatic deletion partially protects against cartilage degeneration. We test whether by becoming senescent, the mesenchymal stromal/stem cells (MSCs), found in the synovial tissue and sub-chondral bone marrow, can contribute to OA development. We established an in vitro p16INK4a-positive senescence model on human MSCs. Upon senescence induction, their intrinsic stem cell properties are altered. When co-cultured with OA chondrocytes, senescent MSC show also a seno-suppressive properties impairment favoring tissue degeneration. To evaluate in vivo the effects of p16INK4a-senescent MSC on healthy cartilage, we rely on the SAMP8 mouse model of accelerated senescence that develops spontaneous OA. MSCs isolated from these mice expressed p16INK4a. Intra-articular injection in 2-month-old C57BL/6JRj male mice of SAMP8-derived MSCs was sufficient to induce articular cartilage breakdown. Our findings reveal that senescent p16INK4a-positive MSCs contribute to joint alteration.